NEW MODELS OF OCULAR INFLAMMATION AND NEW WAYS TO STUDY THEM: (1) Birdshot chorioretinopathy (BC) is strongly associated with HLA-A29. Association between HLA-A29 and uveitis is being studied using HLA-A29 transgenic mice. A strain developed using a construct obtained from French collaborators expressed pathology that turned out to be unrelated to HLA-A29, and instead revealed that the C57BL/6N strain carries the rd8 mutation a finding of great interest for the eye research community. After breeding out the mutation, pathology is not apparent. We now derived a new HLA-A29 construct and produced new founders, which are being followed up for pathology. (2) We continue to collaborate with the group of Dr. Warren Strober (NIAID) on ocular inflammation in NLRP3 knock-in mice. This gene is associated with Muckle-Wells syndrome, which among other pathologies is also associated with inflammation at the ocular surface. Studies with these mice indicate existence of an abnormal response to what we believe to be the normal flora of the ocular surface. These studies may provide new insights into ocular involvement in Muckle-Wells syndrome. (3) Recently initiated studies of mucosal immunology of the ocular surface reveal that the conjunctiva-associated lymphoid tissue (CALT) contains a variety of leukocytes, among them a functionally important population of recently defined innate-like lymphocytes (ILCs). This is a novel finding in what is an understudied area of research. We are currently defining the functionality of these cells in responding to and limiting infection at the ocular surface. The insights may have implications on treatment of conjunctivitis. (3) We have developed three lines of T cell receptor (TCR) Tg mice that express different copy numbers of a TCR specific to the major epitope of IRBP, named R161H, R161M and R161L (suffix stands for high, medium and low TCR expression). R161H and R161M mice develop spontaneous uveitis by 2-3 months of age, whereas the R161L mice only develop disease upon environmental perturbation. These mice allow to study natural triggers of the disease and also as a source of retina-specific T cells, allowing for the first time to study their development and function. (Horai et al., J. Autoimmun. 2013) FUNDAMENTAL MECHANISMS IN TOLERANCE, IMMUNITY AND AUTOIMMUNITY TO RETINAL ANTIGENS (1) Recent findings in R161H mice treated with antibiotics or made germ-free indicated that commensal flora contributes to development of spontaneous uveitis. Mechanistic studies revealed that signaling through the clonotypic TCR in the gut by a non-cognate antigen derived from gut microflora is a necessary (though possibly not sufficient) stimulus (Horai et al, manuscript in preparation). We are currently attempting to dissect which component(s) of the flora is(are) responsible. (2) crossing R161H mice to IFN-g deficient or IL-17 deficient mice unexpectedly revealed a major role for IFN-g in the spontaneous disease. IFN-g-/- R161H mice, but not IL-17-/- R161H mice, had severely reduced spontaneous uveitis scores. These findings raise the possibility that IL-17 produced by R161H cells in the gut, as described above, is a marker of the pathogenic cells but not necessarily the pathogenic cytokine itself. (3) Vitamin (VitA) derivatives are necessary for functional activation of immune cells (published literature). We previously demonstrated the importance of Vitamin A (VitA) and its metabolite, retinoic acid, in ocular immune privilege. Using mice made VitA deficient (VAD), we are studying the role of VitA in regulation of autoimmunity to retina. We found that T cell effector function that was acquired before onset of VAD is fully maintained in the VAD host. These findings may have clinical implications in geographical regions where dietary VitA is limiting. (Horai, Zhou et al, in preparation) (4) We previously demonstrated that autoimmunity to retina could be either Th17 or Th1 driven. Because immune responses have inherent plasticity, targeting only one response could drive the response towards the other, while continuing to fuel pathology. IL27p28 (binds to gp130, a component of both Th17-promoting IL-6, and Th1-promoting IL-27 receptors) was able to inhibit both responses concurrently, by affecting effector priming and lineage stability. Furthermore, similar effects of IL-27p28 in vitro are seen on human T cells. This could point to IL-27 and/or its subunits as a therapeutic mechanism in immunologically complex diseases such as uveitis. (Chong et al, J Autoimmun. 2014). (5) IL-22 has been reported to have both pro-inflammatory and protective effects, depending on the tissue and the model. We used IL-22 and IL-22-receptor deficient mice and anti-IL-22 antibodies to examine effects of IL-22 modulation on EAU. Our data suggest that IL-22 has a local anti-inflammatory and tissue-protective role in the eye (Mattapallil et al., in preparation). (6) We examined the role of T regulatory (Treg) cells in limiting EAU. Data obtained in the induced EAU model in WT mice suggest that Tregs found in uveitic eyes are (i) IRBP specific, (ii) functionally suppressive, and (iii) may play a role in natural resolution of disease. The T cell infiltrate in uveitic eyes of mice that have a polyclonal T cell repertoire is highly enriched in IRBP-specific T cells, both Teff and Treg which appear unimpaired functionally. Finally, FoxP3+ Treg play a role in the natural resolution of EAU and in the maintenance of remission, at least in part due to local effects within the eye (Silver et al, submitted). (7) In chronically inflamed eyes of R161H mice we identified structures resembling tertiary lymphoid tissue. These structures are being studied for their cellular composition, evidence of local T cell priming, and their effects on the course of disease. (J. Kielczewski, in preparation). (8) Self reactivity has been proposed to have a role in neuroprotection, based on studies with adoptively transferred effector T cells (beneficial autoimmunity). R161H mice and their cells provide a way to examine this in a natural setting, where the autoreactive T cells are not initially primed and/or are endogenous to the host. Our data confirm existence of a neuroprotective effect, but it is minor and temporary. Based on our models, a significant neuroprotective role of beneficial autoimmunity in the retina is in question. EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: The innate immune response directly affects immunopathogenic processes and also impacts on adaptive immunity. (1) We previously identified a population of NKT cells that produce IL-17 independently of IL-6 and IL-21 (NKT17). Recent data indicate that IL-17 production in NKT cells and in some other populations of innate lymphoid cells, may be produced via a unique signaling pathway that bypasses STAT3. In vivo experiments suggest that this pathway may have a function in protecting from infections at the ocular surface. (St.Leger, Hansen et al, in preparation). (2) The cytokine IFN-&#947; has both protective and proinflammatory in autoimmunity, which is an unsolved paradox. We uncovered a novel endogenous regulatory circuit based on crosstalk between NK cells and DCs, in which IFN-&#947; and IL-27 are produced as a self-amplifying regulatory feedback loop and are ultimately controlled by IL-10 produced by the interacting DC-NK cells themselves. The IL-27 from this interaction meanwhile dampens the adaptive response and limits autoimmunity by inducing regulatory T cells that control uveitogenic Th17 cells and disease by producing IL-10. NK-DC crosstalk may also limit adaptive immunity in responses to vaccines, and explain the inhibitory effect of CD56-birght NK cells in patients treated for autoimmune disease with Daclizumab therapy (Chong et al, submitted).